CDK5-USP30 signaling pathway regulates MAVS-mediated inflammation via suppressing mitophagy in MPTP/MPP+ PD model.

The discovery of MPTP, an industrial chemical and contaminant of illicit narcotics, which causes parkinsonism in humans, non-human primates and rodents, has led to environmental pollutants exposure being convicted as key candidate in Parkinson's disease (PD) pathogenesis. Though MPTP-induced mitochondrial dysfunction and neuroinflammation are mainly responsible for the causative issue of MPTP neurotoxicity, the underlying mechanism involved remains unclear. Here, we reveal a novel signaling mechanism of CDK5-USP30-MAVS regulating MPTP/MPP+ induced PD. MPP+ (the toxic metabolite of MPTP) treatment not only led to the increased protein levels of USP30 but also to mitophagy inhibition, mitochondrial dysfunction, and MAVS-mediated inflammation in BV2 microglial cells. Both mitophagy stimulation (Urolithin A administration) and USP30 knockdown relieved MAVS-mediated inflammation via restoring mitophagy and mitochondrial function in MPP+-induced cell model. Notably, MPTP/MPP+-induced CDK5 activation regulated USP30 phosphorylation at serine 216 to stabilize USP30. Moreover, CDK5-USP30 pathway promoted MAVS-mediated inflammation in MPTP/MPP+-induced PD model. Inhibition of CDK5 not only had a protective effect on MPP+-induced cell model of PD via suppressing the upregulation of USP30 and the activation of MAVS inflammation pathway in vitro, but also prevented neurodegeneration in vivo and alleviated movement impairment in MPTP mouse model of PD. Overall, our study reveal that CDK5 blocks mitophagy through phosphorylating USP30 and activates MAVS inflammation pathway in MPTP/MPP+-induced PD model, which suggests that CDK5-USP30-MAVS signaling pathway represents a valuable treatment strategy for PD induced by environmental neurotoxic pollutants in relation to MPTP.

Increased release of microvesicles containing mitochondria is associated with the myeloid differentiation of AML-M5 leukaemia cells.

Extensive literature has demonstrated that acute myeloid leukaemia (AML) cells show enhanced mitochondrial biogenesis and increased reliance on oxidative phosphorylation (OXPHOS) compared with normal hematopoietic progenitors, and one hallmark of AML leukaemia blasts is myeloid differentiation blockade. However, relatively few reports have linked these processes. Recent studies have indicated that therapies that overcome differentiation arrest represent an effective treatment strategy. Here, we identified that the disruption of the mitochondrial mass and energy metabolism promotes leukaemia cellular myeloid differentiation. In this study, we showed that acute monocytic leukaemia (AML-M5) cells package mitochondria in microvesicles (MVs) when MVs shed from membranes. Additionally, during myeloid differentiation, we report for the first time that differentiated leukaemia cells release more MVs than undifferentiated leukaemia cells. Targeting the formation of MVs using a specific inhibitor (Y-27632) restrained myeloid differentiation, suggesting that the increased release level of MVs plays an important role in regulating myeloid differentiation. Furthermore, the intracellular mitochondria and ATP levels were decreased after leukaemia cells overcame the differentiation blockade. Moreover, rotenone, which is used to inhibit the respiratory chain and ATP production, had a strong effect on myeloid differentiation in monocytic leukaemia cells. Collectively, these studies uncovered the relationship between mitochondrial function and myeloid differentiation and may provide more insight into the diagnosis and treatment of AML.

Identification of an alternative ligand-binding pocket in peroxisome proliferator-activated receptor gamma and its correlated selective agonist for promoting beige adipocyte differentiation.

The pharmacological activation of peroxisome proliferator-activated receptor gamma (PPARγ) is a convenient and promising strategy for promoting beige adipocyte biogenesis to combat obesity-related metabolic disorders. However, thiazolidinediones (TZDs), the full agonists of PPARγ exhibit severe side effects in animal models and in clinical settings. Therefore, the development of efficient and safe PPARγ modulators for the treatment of metabolic diseases is emerging. In this study, using comprehensive methods, we report a previously unidentified ligand-binding pocket (LBP) in PPARγ and link it to beige adipocyte differentiation. Further virtual screening of 4097 natural compounds based on this novel LBP revealed that saikosaponin A (NJT-2), a terpenoid compound, can bind to PPARγ to induce coactivator recruitment and effectively activate PPARγ-mediated transcription of the beige adipocyte program. In a mouse model, NJT-2 administration efficiently promoted beige adipocyte biogenesis and improved obesity-associated metabolic dysfunction, with significantly fewer adverse effects than those observed with TZD. Our results not only provide an advanced molecular insight into the structural ligand-binding details in PPARγ, but also develop a linked selective and safe agonist for obesity treatment.

Blockage of PPARγ T166 phosphorylation enhances the inducibility of beige adipocytes and improves metabolic dysfunctions.

Beige adipocytes in mammalian white adipose tissue (WAT) can reinforce fat catabolism and energy expenditure. Promoting beige adipocyte biogenesis is a tantalizing tactic for combating obesity and its associated metabolic disorders. Here, we report that a previously unidentified phosphorylation pattern (Thr166) in the DNA-binding domain of PPARγ regulates the inducibility of beige adipocytes. This unique posttranslational modification (PTM) pattern influences allosteric communication between PPARγ and DNA or coactivators, which impedes the PPARγ-mediated transactivation of beige cell-related gene expression in WAT. The genetic mutation mimicking T166 phosphorylation (p-T166) hinders the inducibility of beige adipocytes. In contrast, genetic or chemical intervention in this PTM pattern favors beige cell formation. Moreover, inhibition of p-T166 attenuates metabolic dysfunction in obese mice. Our results uncover a mechanism involved in beige cell fate determination. Moreover, our discoveries provide a promising strategy for guiding the development of novel PPARγ agonists for the treatment of obesity and related metabolic disorders.

A novel mesenchymal stem cell-based regimen for acute myeloid leukemia differentiation therapy.

Currently the main treatment of acute myeloid leukemia (AML) is chemotherapy combining hematopoietic stem cell transplantation. However, the unbearable side effect of chemotherapy and the high risk of life-threatening infections and disease relapse following hematopoietic stem cell transplantation restrict its application in clinical practice. Thus, there is an urgent need to develop alternative therapeutic tactics with significant efficacy and attenuated adverse effects. Here, we revealed that umbilical cord-derived mesenchymal stem cells (UC-MSC) efficiently induced AML cell differentiation by shuttling the neutrophil elastase (NE)-packaged extracellular vesicles (EVs) into AML cells. Interestingly, the generation and release of NE-packaged EVs could be dramatically increased by vitamin D receptor (VDR) activation in UC-MSC. Chemical activation of VDR by using its agonist 1α,25-dihydroxyvitamin D3 efficiently enhanced the pro-differentiation capacity of UC-MSC and then alleviated malignant burden in AML mouse model. Based on these discoveries, to evade the risk of hypercalcemia, we synthetized and identified sw-22, a novel non-steroidal VDR agonist, which exerted a synergistic pro-differentiation function with UC-MSC on mitigating the progress of AML. Collectively, our findings provided a non-gene editing MSC-based therapeutic regimen to overcome the differentiation blockade in AML.

Crosstalk between IL-15Rα+ tumor-associated macrophages and breast cancer cells reduces CD8+ T cell recruitment.

BACKGROUND: Interleukin-15 (IL-15) is a promising immunotherapeutic agent owing to its powerful immune-activating effects. However, the clinical benefits of these treatments are limited. Crosstalk between tumor cells and immune cells plays an important role in immune escape and immunotherapy drug resistance. Herein, this study aimed to obtain in-depth understanding of crosstalk in the tumor microenvironment for providing potential therapeutic strategies to prevent tumor progression. METHODS: T-cell killing assays and co-culture models were developed to determine the role of crosstalk between macrophages and tumor cells in breast cancer resistant to IL-15. Western blotting, histological analysis, CRISPR-Cas9 knockout, multi-parameter flow cytometry, and tumor cell-macrophage co-injection mouse models were developed to examine the mechanism by which IL-15Rα+ tumor-associated macrophages (TAMs) regulate breast cancer cell resistance to IL-15. RESULTS: We found that macrophages contributed to the resistance of tumor cells to IL-15, and tumor cells induced macrophages to express high levels of the α subunit of the IL-15 receptor (IL-15Rα). Further investigation showed that IL-15Rα+ TAMs reduced the protein levels of chemokine CX3C chemokine ligand 1 (CX3CL1) in tumor cells to inhibit the recruitment of CD8+ T cells by releasing the IL-15/IL-15Rα complex (IL-15Rc). Administration of an IL-15Rc blocking peptide markedly suppressed breast tumor growth and overcame the resistance of cancer cells to anti- programmed cell death protein 1 (PD-1) antibody immunotherapy. Interestingly, Granulocyte-macrophage colony-stimulating factor (GMCSF) induced γ chain (γc) expression to promote tumor cell-macrophage crosstalk, which facilitated tumor resistance to IL-15. Additionally, we observed that the non-transcriptional regulatory function of hypoxia inducible factor-1alpha (HIF-1α) was essential for IL-15Rc to regulate CX3CL1 expression in tumor cells. CONCLUSIONS: The IL-15Rc-HIF-1α-CX3CL1 signaling pathway serves as a crosstalk between macrophages and tumor cells in the tumor microenvironment of breast cancer. Targeting this pathway may provide a potential therapeutic strategy for enhancing the efficacy of cancer immunotherapy.

Triple signal amplification strategy for the ultrasensitive electrochemical detection of human papillomavirus 16 E6/E7 mRNA.

Human papilloma virus (HPV) is the primary causative agent of cervical, vaginal, and vulvar cancers. HPV E6/E7 mRNA detection has been proven to improve the specificity and positive predictive value compared with HPV DNA testing in screening, whereby, it may possess higher diagnostic potential. Herein, to establish the ultrasensitive and specific detection of HPV E6/E7 mRNA, we developed a novel triple signal amplification strategy, combined with gold nanoparticles (AuNPs), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and high affinity biotin-avidin system. This novel proposed signal amplification strategy exhibits the desired detection limit of 0.08 fM (approximately 100 copies) and a wide linear range from 0.1 pmol/mL to 100 nmol/mL for HPV16 E6/E7 mRNA detection. Importantly, the present novel biosensor is 10-100 times more sensitive than conventional RT-PCR in detecting HPV16 E6/E7 mRNA positive clinical samples. Conclusively, this biosensor shows good stability, selectivity, and reproducibility, which demonstrates its potential in future clinical diagnosis with desirable sensitivity and specificity.

Establishment of a novel mesenchymal stem cell-based regimen for chronic myeloid leukemia differentiation therapy.

Chronic myeloid leukemia (CML) is characterized by the accumulation of malignant and immature white blood cells which spread to the peripheral blood and other tissues/organs. Despite the fact that current tyrosine kinase inhibitors (TKIs) are capable of achieving the complete remission by reducing the tumor burden, severe adverse effects often occur in CML patients treated with TKIs. The differentiation therapy exhibits therapeutic potential to improve cure rates in leukemia, as evidenced by the striking success of all-trans-retinoic acid in acute promyelocytic leukemia treatment. However, there is still a lack of efficient differentiation therapy strategy in CML. Here we showed that MPL, which encodes the thrombopoietin receptor driving the development of hematopoietic stem/progenitor cells, decreased along with the progression of CML. We first elucidated that MPL signaling blockade impeded the megakaryocytic differentiation and contributed to the progression of CML. While allogeneic human umbilical cord-derived mesenchymal stem cells (UC-MSCs) treatment efficiently promoted megakaryocytic lineage differentiation of CML cells through restoring the MPL expression and activating MPL signaling. UC-MSCs in combination with eltrombopag, a non-peptide MPL agonist, further activated JAK/STAT and MAPK signaling pathways through MPL and exerted a synergetic effect on enhancing CML cell differentiation. The established combinational treatment not only markedly reduced the CML burden but also significantly eliminated CML cells in a xenograft CML model. We provided a new molecular insight of thrombopoietin (TPO) and MPL signaling in MSCs-mediated megakaryocytic differentiation of CML cells. Furthermore, a novel anti-CML treatment regimen that uses the combination of UC-MSCs and eltrombopag shows therapeutic potential to overcome the differentiation blockade in CML.

B7-H3 augments the pro-angiogenic function of tumor-associated macrophages and acts as a novel adjuvant target for triple-negative breast cancer therapy.

B7-H3 is an immune checkpoint molecule from the B7 superfamily. It has been widely studied in tumor immune evasion in certain types of cancer. In our preliminary study, we found that B7-H3 is specifically enriched in tumor-associated macrophages (TAMs) in triple-negative breast cancer (TNBC) patients and strongly correlated with poor clinical prognosis. However, the role of B7-H3 in breast cancer remains elusive. Our current study aims to explore the potential of B7-H3 as a novel target in TNBC therapy. Here, we demonstrated that B7-H3 enriched on TAMs is tightly correlated with TNBC clinical progression. B7-H3high TAMs exhibit great pro-metastatic and immunosuppressive functions by intriguing extracellular matrix (ECM) reconstruction and tumor angiogenesis, therefore helping tumor cell dissemination and dampening T-cell infiltration in tumor microenvironment (TME). Importantly, targeting blockade of B7-H3 by anti-B7-H3 antibody improves the tumor vasculature disorder, thereby enhancing chemotherapy and PD-1 therapy efficacy. In conclusion, our study establishes the correlation between B7-H3high TAMs and TNBC progression for the first time. By exploring the possibility of targeting B7-H3 expressed in both tumor cells and TAMs, we suggest that B7-H3 could be a promising target in clinical TNBC treatment.

Peroxisome proliferator-activated receptor gamma (PPARγ) activation and metabolism disturbance induced by bisphenol A and its replacement analog bisphenol S using in vitro macrophages and in vivo mouse models.

Bisphenol A (BPA) and its replacement analog, bisphenol S (BPS), have been proposed as environmental obesogen to disrupt the lipid metabolism through regulating peroxisome proliferator-activated receptor gamma (PPARγ) receptor. However, there is a dearth of information on whether this biological effect can occur in human macrophage, a cell type which closely interacts with adipocytes and hepatocytes to control lipid metabolism. Here, we for the first time investigate the activity of BPA and BPS on PPARγ pathway in human macrophages. The results demonstrated that BPA and BPS served as activators of PPARγ in human macrophage cell line, and significantly induced the expression of lipid metabolism-related genes, including fatty acid binding protein 4 (FABP4), cluster of differentiation 36 (CD36) and nuclear receptor subfamily 1 group H member 3 (NR1H3). In PPARγ knockout cells, expression of these genes was down-regulated, suggesting that these genes are dependent on PPARγ. The underlying mechanisms were further investigated using an in vivo mouse model, and the results confirmed the induction of PPARγ and its respective target genes in mice following exposure to BPA or BPS. Moreover, the observed alteration of PPARγ expression highly correlated with the disturbance of metabolism profiles in liver tissues as detected by 1H Nuclear Magnetic Resonance (NMR)-based metabonomics. Overall, this study provided the first evidence that BPA and BPS activated PPARγ and its target genes in human macrophages, and provided comprehensive information to confirm that BPA and BPS disturb the metabolism through targeting PPARγ via both in vitro assays and in vivo animal models.

CDK5 Inhibition Abrogates TNBC Stem-Cell Property and Enhances Anti-PD-1 Therapy.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, in which the higher frequency of cancer stem cells (CSCs) correlates with the poor clinical outcome. An aberrant activation of CDK5 is found to associate with TNBC progression closely. CDK5 mediates PPARγ phosphorylation at its Ser 273, which induces CD44 isoform switching from CD44s to CD44v, resulting in an increase of stemness of TNBC cells. Blocking CDK5/pho-PPARγ significantly reduces CD44v+ BCSCs population in tumor tissues, thus abrogating metastatic progression in TNBC mouse model. Strikingly, diminishing stemness transformation reverses immunosuppressive microenvironment and enhances anti-PD-1 therapeutic efficacy on TNBC. Mechanistically, CDK5 switches the E3 ubiquitin ligase activity of PPARγ and directly protects ESRP1 from a ubiquitin-dependent proteolysis. This finding firstly indicates that CDK5 blockade can be a potent strategy to diminish stemness transformation and increase the response to PD-1 blockade in TNBC therapy.

Treatment of obesity-related inflammation with a novel synthetic pentacyclic oleanane triterpenoids via modulation of macrophage polarization.

BACKGROUND: Obesity leads to the chronic inflammation in the whole body and triggers the macrophage polarization to the pro-inflammatory phenotype. Targeting macrophage polarization provides a promising therapeutic strategy for obesity-related metabolic disorders and inflammation. Here, we show that SO1989, a derivative of natural occurring compound oleanolic acid, restores the balance between M1-polarized and M2-polarized macrophages in high fat diets (HFD)-induced obese mice resulting in the improvement of adipose inflammation and the metabolic dysfunctions. METHODS: Histological analysis, magnetic cell sorting and FACS, in vitro cell model of adipose inflammation, Western blotting, HFD mice model. FINDINGS: SO1989 exhibits similar or even stronger activity in inhibiting inflammation and M1 polarization of macrophages both in vitro and in vivo compared to its analogue CDDO-Me, previously known as a powerful anti-inflammation chemical small molecule. In addition, SO1989 can significantly increase the level of fatty acid oxidation in macrophages which can efficiently facilitate M2 polarization of macrophages. Unlike CDDO-Me, SO1989 shows less adverse effects on obese mice. INTERPRETATION: Taken all together, our findings identify SO1989 as a modulator in macrophage polarization and a safer potential leading compound for pro-resolution of inflammation treatment in metabolic disorders. FUND: Supported by grants from the National Key Research and Development Plan (2017YFA0506000, 2017YFA0205400) and National Natural Science Foundation of China (81673439) and Natural Science Fund project in Jiangsu Province (BK20161408).

Caspase-1 cleaves PPARγ for potentiating the pro-tumor action of TAMs.

Tumor-associated macrophages are increasingly viewed as a target of great relevance in the tumor microenvironment, because of their important role in cancer progression and metastasis. However, the endogenous regulatory mechanisms underlying tumor-associated macrophage differentiation remain largely unknown. Here, we report that caspase-1 promotes tumor-associated macrophage differentiation by cleaving peroxisome proliferator-activated receptor gamma (PPARγ) at Asp64, thus generating a 41 kDa fragment. This truncated PPARγ translocates to mitochondria, where it directly interacts with medium-chain acyl-CoA dehydrogenase (MCAD). This binding event attenuates MCAD activity and inhibits fatty acid oxidation, thereby leading to the accumulation of lipid droplets and promoting tumor-associated macrophage differentiation. Furthermore, the administration of caspase-1 inhibitors or the infusion of bone marrow-derived macrophages genetically engineered to overexpress murine MCAD markedly suppresses tumor growth. Therefore, targeting the caspase-1/PPARγ/MCAD pathway might be a promising therapeutic approach to prevent tumor progression.Tumor associated macrophages (TAMs) promote cancer progression. Here, the author show that caspase-1 promotes TAMs differentiation by attenuating medium-chain acyl-CoA dehydrogenase activity and that inhibition of this axis results in suppression of tumour growth in a transgenic mouse model of breast cancer.

Activation of PPARγ by a Natural Flavonoid Modulator, Apigenin Ameliorates Obesity-Related Inflammation Via Regulation of Macrophage Polarization.

PPARγ has emerged as a master regulator of macrophage polarization and is the molecular target of the thiazolidinedione drugs. Here we show that apigenin binds and activates PPARγ by acting as a modulator. Activation of PPARγ by apigenin blocks p65 translocation into nuclei through inhibition of p65/PPARγ complex translocation into nuclei, thereby decreasing NF-κB activation and favoringM2 macrophage polarization. In HFD and ob/ob mice, apigenin significantly reverses M1 macrophage into M2 and reduces the infiltration of inflammatory cells in liver and adipose tissues, as well as decreases the levels of pro-inflammatory cytokines, thereby alleviating inflammation. Strikingly, apigenin reduces liver and muscular steatosis, decreases the levels of ALT, AST, TC and TG, improving glucose resistance obviously. Unlike rosiglitazone, apigenin does not cause significant weight gain, osteoporosis et al. Our findings identify apigenin as a modulator of PPARγ and a potential lead compound for treatment of metabolic disorders.

Effect of vitamin C administration on hydrogen peroxide-induced cytotoxicity in periodontal ligament cells.

Periodontitis is a disease, which is associated with chronic inflammation and leads to significant destruction of periodontal tissues. Periodontal ligament cells (PDLCs) constitute the largest cell population in PDL tissues and a considerable body of evidence has demonstrated an association between oxidative stress and the progression of periodontitis. However, the effects on PDLCs exposed to hydrogen peroxide (H2O2) and the molecular mechanisms by which H2O2 affects periodontitis remain to be elucidated. In the present study, the potential cytotoxic effect of H2O2 and the antioxidative function of vitamin C (Vc) in PDLCs were investigated. The results demonstrated that H2O2 treatment decreased the viability of PDLCs. The decreased PDLC viability was primarily induced by apoptosis, which was evidenced by cleaved caspases-3, caspases-9 and poly (ADP-ribose) polymerase. Following optimal Vc addition, the proapoptotic effects of H2O2 were partially antagonized. Taken together, the present study demonstrated that H2O2 primarily induced the apoptosis of PDLCs and that these adverse effects were partially rescued following treatment with Vc. These results revealed how H2O2 promotes the progression of periodontitis and provide an improved understanding of the reversal effect of antioxidant treatment. Therefore, optimal Vc administration may provide a potentially effective technique in periodontal therapy.